Mutation of lipoprotein processing pathway genelspAor inhibition of LspA activity by globomycin increases MRSA resistance to β-lactam antibiotics

Abstract

The Staphylococcus aureus cell envelope comprises numerous components, including peptidoglycan (PG), wall teichoic acids (WTA), lipoteichoic acids (LTA), targeted by antimicrobial drugs. MRSA resistance to methicillin is mediated by the mecA-encoded β-lactam-resistant transpeptidase, penicillin binding protein 2a (PBP2a). However, PBP2a-dependent β-lactam resistance is also modulated by the activity of pathways involved in the regulation or biosynthesis of PG, WTA or LTA. Here, we report that mutation of the lipoprotein signal peptidase II gene, lspA, from the lipoprotein processing pathway, significantly increased β-lactam resistance in MRSA. Mutation of lgt, which encodes diacylglycerol transferase (Lgt) responsible for synthesis of the LspA substrate did not impact beta-lactam susceptibility. Consistent with previous reports, lgt and lspA mutations impaired growth in chemically defined media, but not in complex broth. MRSA exposure to the LspA inhibitor globomycin also increased β-lactam resistance. Mutation of lgt in an lspA background restored β-lactam resistance to wild type. The lspA mutation had no effect on PBP2a expression, PG composition or autolytic activity indicating a potential role for WTA or LTA. The lspA and lgt mutants exhibited marginally increased resistance to the D-alanine pathway inhibitor D-cycloserine. In addition, mutation of lgt and multicopy lspA expression, but not mutation of lspA, significantly increased susceptibility to the lipoteichoic acid synthase inhibitor Congo red revealing complex interplay between lipoprotein processing mutations and the expression/stability of cell surface glycopolymers. These findings indicate that accumulation of the LspA substrate, diacylglyceryl lipoprotein, increases MRSA resistance to β-lactam antibiotics through impacts on cell envelope components other than PG.

Accumulation of Succinyl Coenzyme A Perturbs the Methicillin-Resistant Staphylococcus aureus (MRSA) Succinylome and Is Associated with Increased Susceptibility to Beta-Lactam Antibiotics

“…PG was extracted from the samples as described previously ( 49 , 78 ). This analysis of wild-type JE2 PG was also used as a part of a separate study ( 67 ) and is reported again here for comparison to NE569 and sucC suppressor 1. Mass spectrometry (MS) was performed on a Waters XevoG2-XS QTof mass spectrometer.…”

“…Genomic DNA extraction, sequencing by MicrobesNG ( http://www.microbesng.uk ), and analysis were performed as described previously ( 67 ). Wild-type JE2 reads were aligned against the published USA300 FPR3757 genome sequence (RefSeq accession number NC_007793.1 ) and assembled into a contig.…”

“…Phage 80α was used to transduce the transposon insertion from NE569 into JE2, DAR173, and USA300 to ensure that background mutations were not responsible for the antibiotic resistance phenotype, as described previously ( 67 ). Transposon insertions were verified using PCR amplification of target loci.…”

“…Overnight MHB cultures were used to inoculate 25 ml of MHB plus 2% NaCl, with or without 0.5 µg/ml oxacillin, to a starting OD 600 of 0.05, followed by incubation at 35°C (with 200-rpm shaking) until an OD 600 of 0.8 was reached. The cells were then pelleted and resuspended in PBS to an OD 600 of 10, and PBP2a Western blot analyses were performed as described previously ( 67 ). Three independent experiments were performed and a representative blot presented.…”

Purine nucleosides interfere with c-di-AMP levels and act as adjuvants to re-sensitise MRSA to β-lactam antibiotics

“…Genetic manipulation of S. aureus. Phage 80a was used as described previously (70) to transduce the transposon insertions from NE650, NE1419 into the parent JE2 strain to rule out the possibility that uncharacterised mutations in the NTML mutants may contribute to the increased antibiotic resistance phenotypes. Transposon insertions were verified by PCR amplification of the target loci using primers listed in Table S2.…”